CN105543335A - Measurement method of acid phosphatase activity - Google Patents
Measurement method of acid phosphatase activity Download PDFInfo
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- CN105543335A CN105543335A CN201610013539.XA CN201610013539A CN105543335A CN 105543335 A CN105543335 A CN 105543335A CN 201610013539 A CN201610013539 A CN 201610013539A CN 105543335 A CN105543335 A CN 105543335A
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- acid phosphatase
- activity
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
- C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
Abstract
The invention discloses a measurement method of acid phosphatase activity. According to the method, L-ascorbic acid-2-phosphoric acid serves as a catalytic substrate of acid phosphatase, the substrate is hydrolyzed into L-ascorbic acid under catalysis of acid phosphatase, because bivalent copper can be reduced into monovalent copper by L-ascorbic acid so that a bivalent copper compound serving as a color developing probe can be converted into a monovalent copper compound in deep color, the higher the activity of acid phosphatase in samples is, the more obvious the color change is, and therefore semi-quantitative colorimetric analysis of the activity of acid phosphatase is achieved, or quantitative detection can be performed with an ultraviolet-visible spectrophotometer. By means of the method, acid phosphatase with the concentration of 0.48 mU.mL<-1> can be detected. The method is high in sensitivity, good in selectivity, high in anti-interference capacity, good in repeatability, easy to operate, short in detection time and low in cost, can be used for quantitatively measuring the activity of acid phosphatase in serum samples, and a fast, simple, convenient, low-cost, sensitive and stable-performance colorimetric analysis method is provided for detecting the activity of acid phosphatase in clinical samples.
Description
Technical field
The invention belongs to bioassay technique field, be specifically related to a kind of measuring method of activity of acid phosphatase, particularly relate to a kind of method based on colorimetric analysis Fast Measurement activity of acid phosphatase.
Background technology
Acid phosphatase (acidphosphatase, EC3.1.3.2, ACP) is mainly present in scavenger cell, is positioned in lysosome, and the hydrolysis reaction of energy catalytic phosphatase monoesters, produces inorganic phosphate and corresponding alcohol, phenol or sugar etc.ACP in normal human serum is mainly derived from the tissues such as prostate gland, bone, liver,kidney,spleen, pancreas, no matter men and women, old and young, its content is roughly the same.And Patients of Prostatic Diseases and when there is the diseases such as deformability osteomyelitis, osteogenesis imperfecta, richets, osteosarcoma, multiple myeloma, primary bone tumor, hyperthyroidism, leukemia, mammary cancer, myocardial infarction, hepatitis, liver cirrhosis, cholecystitis, hemolytic disease and acute urinary retention, in serum, the level of ACP all can obviously raise.Such as, clinical study shows, in patients with prostate cancer body, ACP level obviously raises, and the state of an illness of its elevated-levels and prostate cancer is parallel relation substantially: when sb.'s illness took a favorable turn, and ACP level progressively reduces; When again raising, normal prompting cancer has Preventive and prognosis mala.As can be seen here, how simply, the activity of ACP is measured effectively and quickly, especially the activity of ACP in the clinical tissue of complicated component and serum sample, to the auxiliary diagnosis of prostate cancer, especially to the diagnosis of advanced prostate cancer, observation of curative effect and Prognosis scoveillance, there is important clinical value and application prospect widely.
At present, the method measuring ACP activity mainly comprises following several: (1) 4-NPP method: in acid condition, 4-NPP generates yellow water-soluble products p-NP under the katalysis of ACP, this product has photoabsorption at 405nm place, by measuring its absorbance, and compare with typical curve, quantitative analysis can be carried out to ACP activity; (2) Gomori lead nitrate method: in acid condition, take sodium β-glycerophosphate as substrate, discharges phosphoric acid by ACP catalytic hydrolysis, and phosphoric acid is combined with lead nitrate and forms colourless lead phosphate and precipitate, and forms the lead sulfide precipitation of brownish black through ammonium sulfide process; (3) disodium phenyl phosphate method: in acid condition, ACP is hydrolyzed disodium phenyl phosphate and produces free phenol and phosphoric acid, phenol in basic solution with 4-AA effect, through the Tripotassium iron hexacyanide oxidation formed red quinones, its shade is directly proportional to ACP activity.As can be seen here, in clinical detection, the depth of method mainly based on solution colour measuring ACP activity changes (ie in solution is to the selective absorbing of light) carries out quantitative assay colorimetric analysis to substances content, this be due to colorimetric analysis have that equipment is simple, analysis speed is fast, accuracy is high, the good characteristic such as favorable reproducibility and applicable batch samples.But, the shortcomings such as it is low that above-mentioned several method all has sensitivity, poor anti jamming capability, and testing process is loaded down with trivial details.
Summary of the invention
The object of the present invention is to provide a kind of measuring method of activity of acid phosphatase, particularly a kind of method measuring activity of acid phosphatase based on colorimetric analysis.Method of the present invention is highly sensitive, selectivity is good, immunity from interference is strong, reproducible, simple to operate, detection time is short, with low cost, can be used for the quantitative assay of activity of acid phosphatase in serum sample, for the detection of activity of acid phosphatase in clinical sample provides a kind of quick, easy, cheap, sensitive and colorimetric methods of stable performance.
For achieving the above object, the present invention adopts following technical solution:
A kind of measuring method of activity of acid phosphatase, comprise the following steps: after the damping fluid containing L-AA-2-phosphoric acid, bivalent cupric ion, water-soluble phenanthroline derivative and acid phosphatase enzyme solution to be measured are hatched, sxemiquantitative colorimetric detection is carried out to activity of acid phosphatase in sample according to detecting the colour-change of liquid or carries out detection by quantitative with ultraviolet-visible spectrophotometer.
Described damping fluid is the glycine-HCI damping fluid of 50mMpH5.7.
In described detection liquid, the concentration of L-AA-2-phosphoric acid is 50nM ~ 100mM, is preferably 10mM.
In described detection liquid, the concentration of bivalent cupric ion is 5nM ~ 10mM, is preferably 0.2mM.
Described water-soluble phenanthroline derivative comprises bathophenanthroline disulfonic acid salt, bath ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate etc.
In described detection liquid, the concentration of water-soluble phenanthroline derivative is 0.01 ~ 20mM, is preferably 0.4mM.
Described incubation temperature is 20 ~ 37 DEG C, is preferably 25 DEG C.
Described incubation time is 0.5 ~ 150min, is preferably 15min.
Compared with prior art, beneficial effect of the present invention is:
1. method of the present invention is limited to 0.48mUmL to detecting of activity of acid phosphatase
-1, have highly sensitive, selectivity is good, and immunity from interference is strong, reproducible, simple to operate, the advantages such as detection time is short and with low cost;
2. plant and instrument universalness degree required for the present invention is high, and required reagent is commercially produced product, to operator without special technical requirement, and can realize sxemiquantitative colorimetric analysis;
3. method of the present invention can be used for quick, the highly sensitive detection of activity of acid phosphatase in clinical sample, and can greatly reduce testing cost and testing cost.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of the measuring method of activity of acid phosphatase of the present invention.
Fig. 2 is feasibility analysis and the photo of the inventive method colorimetric analysis activity of acid phosphatase.Wherein, A is that all reagent all adds, and B is not Plus acidic Phosphoric acid esterase, and C is not for add L-AA-2-phosphoric acid, and D is not for adding bath ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate-bivalent cupric ion.
Fig. 3 is medium ultraviolet-visible absorption spectra of the present invention with the change curve of activity of acid phosphatase and photo (A) and standard working curve (B).
Fig. 4 is selectivity analysis and the photo of the inventive method colorimetric analysis activity of acid phosphatase.Wherein, A is acid phosphatase, and B is bovine serum albumin, and C is oxyphorase, and D is horseradish peroxidase, and E is chain enzyme avidin, and F is zymoplasm, and G is trypsinase, and H is contrast.
Fig. 5 is the analytical characteristics of the inventive method colorimetric analysis activity of acid phosphatase in serum sample and photo.Wherein, A is in glycine-HCI damping fluid, and B is in 10% normal human sera samples, and C is in 5% normal human sera samples.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, it will be understood to those of skill in the art that following examples are only the preferred embodiments of the present invention, so that understand the present invention better, thus should not be considered as limiting scope of the present invention.
Fig. 1 shows the principle of the measuring method of a kind of activity of acid phosphatase of the present invention, this Cleaning Principle (to bathe ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate) specific as follows:
Acid phosphatase enzymatic hydrolysis L-AA-2-phosphoric acid generates L-AA, bivalent cupric ion is reduced into univalent copper ion by L-AA, make cupric-bath ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate mixture be converted into monovalence copper-bath ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate mixture, thus cause the change of solution colour and absorbancy.Because cupric-bath ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate complex solution is faint yellow, monovalence copper-bath ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate complex solution is brown color, by observing solution colour change, can carry out sxemiquantitative colorimetric analysis to activity of acid phosphatase in sample.In addition, because monovalence copper-bath ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate mixture is at the new absorption peak of 484nm place generation one, its intensity is directly proportional to the concentration of monovalence copper-bath ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate mixture, therefore carries out detection by quantitative by ultraviolet-visible spectrophotometer to activity of acid phosphatase in sample.The sensing range of method of the present invention to activity of acid phosphatase is 0 ~ 100mUmL
-1, detect and be limited to 0.48mUmL
-1.Method of the present invention is highly sensitive, selectivity is good, immunity from interference is strong, reproducible, simple to operate, detection time short (~ 15min), with low cost, can be used for the quantitative assay of activity of acid phosphatase in serum sample, for the detection of activity of acid phosphatase in clinical sample provides a kind of quick, easy, cheap, sensitive and colorimetric methods of stable performance.
Embodiment 1: the feasibility analysis of the inventive method colorimetric analysis activity of acid phosphatase.
By 20 μ L acid phosphatase enzyme solution (100mUmL
-1) and 20 μ LL-xitix-2-phosphoric acid (0.3M), 10 μ L copper sulfate-bath ketone spirit sodium disulfonate mixture (copper sulfate: 12mM, bath ketone spirit sodium disulfonate: 24mM) and 550 μ L glycine-HCI damping fluid (50mM, pH5.7) Homogeneous phase mixing, the mixed solution obtained hatches 15min at 25 DEG C, then the uv-visible absorption spectra of solution within the scope of 800 ~ 300nm is measured also by the colour-change of digital camera recording solution, as shown in Figure 2.Wherein, A is that all reagent all adds, and B is not Plus acidic Phosphoric acid esterase, and C is not for add L-AA-2-phosphoric acid, and D is not for adding copper sulfate-bath ketone spirit sodium disulfonate mixture, and the glycine-HCI damping fluid of the reagent equivalent 50mMpH5.7 do not added substitutes.As can be seen from Figure 2, after 15min, all not there is noticeable change in absorption spectrum and the color of control group solution, experimental group solution then has very strong absorption at 484nm place, and solution colour there occurs significant change.As can be seen here, the method for colorimetric analysis activity of acid phosphatase of the present invention has goodish feasibility.
Embodiment 2: detect the uv-visible absorption spectra of liquid and the relation between colour-change and activity of acid phosphatase.
By 0,10 of 20 μ L, 20,30,40,50,60,70,80,90 and 100mUmL
-1acid phosphatase enzyme solution respectively with containing 20 μ LL-xitix-2-phosphoric acid (0.3M), 10 μ L copper sulfate-bath ketone spirit sodium disulfonate mixture (copper sulfate: 12mM, bath ketone spirit sodium disulfonate: 24mM) and 550 μ L glycine-HCI damping fluid (50mM, pH5.7) dissolution homogeneity mixing, the mixed solution obtained hatches 15min at 25 DEG C, then the uv-visible absorption spectra of solution within the scope of 800 ~ 300nm is measured also by the colour-change of digital camera recording solution, as shown in Figure 3.As can be seen from Fig. 3 (A), along with the increase of activity of acid phosphatase in sample, detect liquid in the corresponding increase of the absorption value at 484nm place.As can be seen from Fig. 3 (B), with detect the absorption value of liquid at 484nm place to sample in activity of acid phosphatase mapping, can obtain good quantitative relationship, its sensing range is 0 ~ 100mUmL
-1(R
2=0.9997), Monitoring lower-cut is 0.48mUmL
-1.
Embodiment 3: the selectivity analysis of the inventive method colorimetric analysis activity of acid phosphatase.
By 20 μ L1mgmL
-1acid phosphatase (A), bovine serum albumin (B), oxyphorase (C), horseradish peroxidase (D), chain enzyme avidin (E), zymoplasm (F), trypsin G) or 50mMpH5.7 glycine-HCI damping fluid (H) respectively with containing 20 μ LL-xitix-2-phosphoric acid (0.3M), 10 μ L copper sulfate-bath ketone spirit sodium disulfonate mixture (copper sulfate: 12mM, bath ketone spirit sodium disulfonate: 24mM) and 550 μ L glycine-HCI damping fluid (50mM, pH5.7) dissolution homogeneity mixing, the mixed solution obtained hatches 15min at 25 DEG C, then the absorption value of solution at 484nm place is measured also by the colour-change of digital camera recording solution, as shown in Figure 4.As can be seen from the figure, except acid phosphatase, other absorption values disturbing albumen that detection liquid all can not be caused at 484nm place or color occur obviously to change.As can be seen here, the method for colorimetric analysis activity of acid phosphatase of the present invention has good selectivity.
Embodiment 4: the analytical characteristics of method in serum sample of colorimetric analysis activity of acid phosphatase of the present invention.
By 20 μ L acid phosphatase enzyme solution (100mUmL
-1) and 20 μ LL-xitix-2-phosphoric acid (0.3M), 10 μ L copper sulfate-bath ketone spirit sodium disulfonate mixture (copper sulfate: 12mM, bath ketone spirit sodium disulfonate: 24mM) and 550 μ L glycine-HCI damping fluid (50mM, pH5.7) Homogeneous phase mixing, the mixed solution obtained hatches 15min at 25 DEG C, then the absorption value of solution at 484nm place is measured also by the colour-change of digital camera recording solution, as shown in Figure 5.Wherein, A is in the glycine-HCI damping fluid of 50mMpH5.7, and B is in 10% normal human sera samples, and C is in 5% normal human sera samples.As can be seen from Figure 5, the inventive method changes in the absorption value at 484nm place and solution colour in serum sample and in damping fluid the acid phosphatase of identical activity does not all have marked difference.As can be seen here, the method for colorimetric analysis activity of acid phosphatase of the present invention can be used for the quantitative assay of activity of acid phosphatase in serum sample, has high clinical value.
Claims (8)
1. the measuring method of an activity of acid phosphatase, it is characterized in that, after damping fluid containing L-AA-2-phosphoric acid, bivalent cupric ion, water-soluble phenanthroline derivative and acid phosphatase enzyme solution to be measured are hatched, sxemiquantitative colorimetric detection is carried out to the activity of acid phosphatase in sample or adopts ultraviolet-visible spectrophotometer to carry out detection by quantitative.
2. the measuring method of activity of acid phosphatase as claimed in claim 1, is characterized in that, described damping fluid adopts the glycine-HCI damping fluid of 50mMpH5.7.
3. the measuring method of activity of acid phosphatase as claimed in claim 1, it is characterized in that, in described detection liquid, the concentration of L-AA-2-phosphoric acid is 50nM ~ 100mM.
4. the measuring method of activity of acid phosphatase as claimed in claim 1, it is characterized in that, in described detection liquid, the concentration of bivalent cupric ion is 5nM ~ 10mM.
5. the measuring method of activity of acid phosphatase as claimed in claim 1, is characterized in that, described water-soluble phenanthroline derivative is bathophenanthroline disulfonic acid salt or bath ketone spirit stilbene-4,4'-bis-(1-azo-3, 4-dihydroxy-benzene)-2,2'-disulfonate.
6. the measuring method of activity of acid phosphatase as claimed in claim 1, it is characterized in that, in described detection liquid, the concentration of water-soluble phenanthroline derivative is 10nM ~ 20mM.
7. the measuring method of activity of acid phosphatase as claimed in claim 1, it is characterized in that, described incubation temperature is 20 ~ 37 DEG C.
8. the measuring method of activity of acid phosphatase as claimed in claim 1, it is characterized in that, described incubation time is 5 ~ 150min.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442484A (en) * | 2016-11-22 | 2017-02-22 | 南京理工大学 | Method for detecting hydrolase activity |
| CN106706578A (en) * | 2016-11-22 | 2017-05-24 | 南京理工大学 | Fluorescence detection method for hydrolase activity |
| CN106769914A (en) * | 2016-11-22 | 2017-05-31 | 南京理工大学 | A kind of method for determining hydrolytic enzyme activities |
| CN113189052A (en) * | 2021-04-14 | 2021-07-30 | 暨南大学 | Acid phosphatase optical fiber biosensor and preparation method and application thereof |
| CN113970592A (en) * | 2020-07-23 | 2022-01-25 | 南京大学 | Mass spectrum sensing chip for quantitative detection of acid phosphatase and preparation method thereof |
Citations (1)
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| CN103649754A (en) * | 2011-07-07 | 2014-03-19 | 杜邦营养生物科学有限公司 | Assay |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103649754A (en) * | 2011-07-07 | 2014-03-19 | 杜邦营养生物科学有限公司 | Assay |
Non-Patent Citations (2)
| Title |
|---|
| GAO Z.等: "High-resolution colorimetric assay for rapid visual readout of phosphatase activity based on gold/silver core/shell nanorod", 《ACS APPL. MATER. INTERFACES.》 * |
| 杨芳芳: "抗坏血酸的测定方法综述", 《甘肃科技纵横》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106442484A (en) * | 2016-11-22 | 2017-02-22 | 南京理工大学 | Method for detecting hydrolase activity |
| CN106706578A (en) * | 2016-11-22 | 2017-05-24 | 南京理工大学 | Fluorescence detection method for hydrolase activity |
| CN106769914A (en) * | 2016-11-22 | 2017-05-31 | 南京理工大学 | A kind of method for determining hydrolytic enzyme activities |
| CN113970592A (en) * | 2020-07-23 | 2022-01-25 | 南京大学 | Mass spectrum sensing chip for quantitative detection of acid phosphatase and preparation method thereof |
| CN113189052A (en) * | 2021-04-14 | 2021-07-30 | 暨南大学 | Acid phosphatase optical fiber biosensor and preparation method and application thereof |
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